fragment (Fc)] to FceRI with high affinity, with thehalf-life of IgE bound to FceRI measured indays (5). Interaction of antigen with FceRI-boundantigen-specific IgE clusters the individual recep-tors (6, 7); this step is required for generation ofintracellular signals that cause mast cells andbasophils to release allergic mediators (3, 8). Theantigen-binding (Fab) portion of FceRI-boundIgE antibodies may differ in their affinity for theantigen [as seen in allergic individuals (9)], pre-sumably affecting the duration of the transmittedsignal and subsequent outcome. Whether FceRIfunctionally distinguishes differences in the affin-ity of IgE antibody and antigen interactions is notclear. To investigate this, we used two previouslydescribed antigens (10): dinitrophenyl-caproate-Fab(DNP, high affinity) and 2-nitrophenyl-caproate-Fab (2NP, low affinity). These differ in their relativeaffinities for binding to FceRI-bound DNP-specificIgE by approximately three orders of magni-tude. In bone marrow–derived mouse mast cells(BMMCs) (11), FceRI phosphorylation was sim-ilar with approximately 100 times as much 2NP(3000 ng/ml) as DNP (30 ng/ml) (fig. S1A); thekinetics of FceRI phosphorylation were un-altered at these concentrations (fig. S1B). How-ever, cellular responses differed as 2NP elicitedless than 20% of the DNP-induced degranulationresponse (Fig. 1A) at 3000 ng/ml and 30 ng/ml,respectively, and showed reduced leukotrieneB4 (Fig. 1B) and cytokine production (Fig. 1C)but enhanced chemokine production (Fig. 1D).DNP- and 2NP-induced responses required thepresence of DNP-specific IgE (fig. S2, A and B),and 2NP treatment had no effect on responsesinitiated through ovalbumin (OVA)–specific IgE(fig. S2, C and D).To explore the differences in DNP- and 2NP-induced FceRI clustering, we used total internalreflection fluorescence (TIRF) microscopy tostudy DNP-specific IgE-bearing mast cells aftertheir contact with either a DNP- or 2NP-imbeddedplanar supported lipid bilayer, while maintainingequal receptor phosphorylation and differencesin mast cell degranulation (fig. S3, A and B).Exposure to DNP resulted in highly mobile re-ceptor clusters that moved from the cell peripherytoward the cell center to form a synapse-like lo-calization as described for the T cell receptor(12, 13) (movie S1). In contrast, treatment with2NP revealed slower movement of receptorclusters and a diffuse distribution with a looselyorganized synapse-like structure at the cell cen-ter (movie S2). Analysis of receptor cluster move-ment with time (Fig. 2A) revealed greater numbersof receptor clusters at the periphery in 2NP-treated cells. The total number of clusters formedat any given time was greater upon DNP treatment(Fig. 2B), but the area occupied by these clusterswas larger after 2NP treatment (Fig. 2C). Therelative mobility of receptor clusters in cells treatedwith 2NP was on average one-third that of clus-ters formed by DNP treatment (Fig. 2D). Phos-photyrosine (a hallmark of intracellular signaling)was localized with both DNP- and 2NP-formedreceptor clusters, although stronger colocalizationwas evident after 2NP treatment (Fig. 2E). Thesefindings demonstrate that the FceRI clusters in-duced by DNP or 2NP differ spatiotemporally.We explored whether differences in the spatio-temporal behavior of receptor clustering by DNP

Fig. 3. Distinct molecular signals are induced in 2NP-treated BMMCs: Enhanced role for Fgr
and LAT2. (A) Phosphorylation of the FceRI (ITAMg Y47) in WT, syk−/−, fyn−/−, and lyn−/− BMMCs
after DNP (30 ng/ml) or 2NP (3000 ng/ml) treatment. Relative increase is normalized to the control
(CTL) for WT. (B) Kinetics of LAT1 and LAT2 phosphorylation detected by Western blot differ in cells
treated with DNP or 2NP. Relative increase is normalized to DNP (0 min). (C) Single cell analysis of
BMMC calcium responses in the absence or presence of extracellular Ca (–[Ca2+]o; +[Ca2+]o) as indicated. Fluorescence intensity of a single cell with time (more than 300 cells were monitored in total)
was measured and shown as the “calcium response” normalized to the baseline fluorescence. Single cells,
solid colored lines; averaged responses, black line with open circles. (D) Localization of the LAT2-RFP
(green) in lat2−/− BMMCs with Alexa Fluor 647–conjugated IgE (red, marking FceRI) after DNP or 2NP
treatment. Images were captured by TIRF microscopy at 120 or 300 s. Fluorescence intensities of IgE-FceRI
microclusters and LAT2-RFP are shown in the graphs. Colocalization was analyzed in 15 to 30 cells for
each condition. ***P < 0.001, Student’s t test. Scale bar, 5 mm. (E) Fluorescence intensities of Lyn-YFP
(green), Fgr-RFP (white), IgE-FceRI microclusters (cyan) in BMMCs derived from fgr−/− hck−/− lyn−/−
mice treated with DNP or 2NP for 60 or 120 s. Mean fluorescence intensity was measured from at least
50 cells for each condition. ***P < 0.001, Student’s t test. Scale bar, 5 mm. (F) CCL2 secretion by BMMCs
from the indicated mice. *P < 0.05, **P < 0.01, ***P < 0.001; one-way ANOVA. Data are means T SE
from at least four individual experiments.